CN107828706A

CN107828706A - A kind of eGFP is marked, anti-swine infectious enterogastritis and the restructuring VREF vaccine strain of pig epidemic diarrhea and its application

Info

Publication number: CN107828706A
Application number: CN201710972807.5A
Authority: CN
Inventors: 徐义刚; 王丽; 乔薪瑗; 唐丽杰; 李经; 李一经; 于美玲
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2017-10-18
Filing date: 2017-10-18
Publication date: 2018-03-23

Abstract

A kind of marked the invention discloses eGFP, anti-swine infectious enterogastritis and the restructuring VREF vaccine strain of pig epidemic diarrhea and its application.Described vaccine strain transmits live vector using the VREF obtained from the separation of chitling road colonic segment as vaccine antigen, wherein the recombinant expression carrier of constitutive expression TGEV S protein D antigen sites and PEDV S protein neutralizing epitopes area ps420 albumen containing eGFP marks.The anti-swine infectious enterogastritis and the divalence oral vaccine of pig epidemic diarrhea being prepared by above-mentioned restructuring VREF vaccine strain can reach primary immune response and prevent the purpose of two kinds of diseases, and have the characteristics that green, highly effective and safe, probiotic good.

Description

The restructuring of a kind of eGFP is marked, anti-swine infectious enterogastritis and pig epidemic diarrhea VREF vaccine strain and its application

Technical field

The present invention relates to a kind of vaccine strain and its construction method, more particularly to one kind being capable of anti-swine infectious enterogastritis simultaneously With the VREF vaccine strain and its construction method of pig epidemic diarrhea, further relate to the vaccine strain and preparing anti-swine infectious stomach and intestine Application in scorching and pig epidemic diarrhea divalence oral vaccine.The invention belongs to veterinary drug technical field.

Background technology

Transmissible gastro-enteritis virus (Transmissible gastroenteritis of swine virus, TGEV) It is that coronavirus genus is viral with Porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus, PEDV), pig Cause after only infecting with chordapsus, diarrhoea and dead transmissible gastroenteritis of swine (TGE) and pig epidemic for cardinal symptom Rush down (PED).TGE and PED outbursts be in obvious seasonal characteristics, and winter-spring season be the multiple phase, and presentation colony is broken out during morbidity Situation, newborn piglet morbidity and mortality can reach 100%, and massive losses are brought to pig industry.Antibiotic and antiviral class This sick DeGrain of routine medication, mainly prevented using vaccine inoculation.At present, the son that domestic and international market uses Pig coronavirus property diarrhoea main 2 class of vaccine：One kind is inactivated vaccine, and country variant develops TGE strains used in inactivated vaccine Difference, such as the U.S. use CKP strains using TGE-Vac strains, Japan using TO-163 strains, Hungary, and China uses magnificent strain；Separately One kind is attenuated vaccine, and Harbin veterinary institute successfully have developed TGEV and PEDV Bi-combined attenuated Vaccines within 2004, existing America and Europe The vaccine that country uses is mainly attenuated vaccine, and commercialization attenuated vaccine mainly has the CV777 strains in China, the P-5V of Japan at present Strain, the KPED-9 strains and DR13 strains of South Korea, TGEV, PEDV, PoRV (G types) trigeminal live vaccine of domestic initiation in 2015 are formal Go into operation and list.

From phase late 1960s, just there is the report on TGE in China, and epidemic-stricken area more expands in recent years, and and pig Epidemic diarrhea, porcine rotavirus disease mixed infection, serious economic loss is caused to pig industry.In by the end of April, 2013, PED is broken out in U.S. swinery, the subsequent disease spreads to more than 20 individual state of the U.S..TGEV is similar with PEDV pathogenesis, TGEV Mainly fallen ill with PEDV through intestinal mucosa infection animal, intestinal villus epithelial cell is TGEV and PEDV target cell.Numerous studies As a result show, piglet mainly obtains source of parents sIgA antibody by colostrum and produces the passive immune protection suffered from diarrhoea to coronavirus property. Therefore, mucosa-immune is the important channel for protecting piglet adaptive immune, and newborn source is produced by body after sow vaccine inoculation SIgA antibody so to suckling pig provide protection, or immune piglet make its own produce mucosal immune response, could be body Enough protections to viral infection resisting are provided.

The sIgA that traditional TGE and PED unit prices seedling and multi-joint inactivated vaccine are unable to stimulating animal body generation sufficient amount resists Body, protecting effect is bad, and seedling cost is also costly.Inactivated vaccine produces immunity time up to two in pig body simultaneously Week, it is necessary to be inoculated with, to ensure preventive effect in advance.Although attenuated vaccine can cause mucosa-immune, due to there is cost it is high, Easy reversion, there is the defects of potential virulence enhancing risk, limit its application in production.Through Houhai acupoint vaccine inoculation, though can Cause mucosal immune response, be the breakthrough to routine immunization approach, enhance immune protective efficiency, be inoculated with once, but During vaccine inoculation, depth of needle need to increase and deepen with pig age, and parallel with rectum or slightly elevated inoculation is kept during inserting needle, is given simultaneously Appropriate Baoding is wanted during in-pig vaccine inoculation, otherwise easily causes mechanicalness to be miscarried.

TGE and PED traditional vaccines existing defects in terms of immunoprotection and security, and recombinant vaccine is because of cost It is low, production is simple, convenient storage and turn into TGE and PED vaccine research focuses.TGEV and PEDV surface spike protein S protein It is the target protein that mediate retroviral is combined with host cell, can induce body and produce neutralizing antibody, be that all kinds of recombinant vaccines are ground The preferred immunizing antigen studied carefully.Meng in 2013 etc. passes through intramuscular injection path using TGEV the and PEDV albumen of eucaryon amalgamation and expression Immune mouse, inducing mouse generate anti-TGEV and PEDV neutralizing antibody.Research on theory and practice shows, with oral vaccination Mode vaccine inoculation can induce body generation and be directed to mucosa infection mucosa-immune antibody sIgA the most direct, be exempted from animal Epidemic disease is protected.Zhang in 2016 etc. to express the restructuring salmonella typhimurium oral immunity pig of TGEV and PEDV S proteins, though So can Induction experiments pig produce neutralizing antibody, but as vaccine antigen transmission carrier, bio-safety to be present hidden by the use of pathogenic bacteria Suffer from.Express as vaccine antigen by using food-grade microorganisms and will more pacify with transmitting carrier development TGE and PED oral vaccines Entirely.Lactic acid bacteria is widely used in food, feed processing industry as the main probiotics in probiotics.

Nineteen ninety Isabel etc. is tested by monoclonal antibody to be proved and positions to complete TGEV S proteins and can stimulate machine Body produces the region of neutralizing antibody, and is divided into four regions, is defined as A, B, C, D antigen site, wherein A, B and D position Point is highly conserved.Jiang in 2016 etc. is that immunizing antigen transmits vector construction table with Lactobacillus casei (L.casei 393) Up to the recombination lactic acid bacteria preparation of TGEV S protein D antigen sites, oral immunity pig generates certain immune protection effectiveness.But should Recombinant lactic acid bacteria system employs chloramphenicol resistance gene and alternatively marked, with the lactobacillus preparation answering in practice With the transfer of resistance factor will bring serious biological safety consequence, while the recombinant lactic acid bacteria system is induction type, lack In the environment of weary derivant, recombinant lactic acid bacteria will stop the secretion of vaccine antigen, is unfavorable for large-scale production, further constrains The popularization and application of the recombination lactic acid bacteria preparation.

It is therefore an object of the present invention to develop more safely, effectively, inexpensively, inoculation can easily make body produce protection Property mucosa-immune reaction TGE and PED bivalent vaccines.

The content of the invention

An object of the present invention be to provide a kind of eGFP mark, can anti-swine infectious enterogastritis and pig be popular simultaneously Property diarrhoea restructuring VREF vaccine strain.

The second object of the present invention is to provide a kind of anti-pig being prepared by above-mentioned restructuring VREF vaccine strain and infected The divalence oral vaccine of property gastroenteritis and pig epidemic diarrhea.

The divalence that the third object of the present invention is to provide described restructuring VREF vaccine strain and its is prepared is oral Application of the vaccine in prevention transmissible gastroenteritis of swine and pig epidemic diarrhea.

In order to achieve the above object, present invention employs following technological means：

The characteristics of present invention mainly falls ill according to TGEV and PEDV through intestinal mucosa infection animal, sets from mucosa-immune angle Meter vaccine is infected with preventing TGEV and PEDV simultaneously, piglet clinic diarrhoeal diseases viral disease is occurred and control it is significant, Be present invention mainly solves key scientific problems.In order to develop, prebiotic performance is good, safe and efficient anti-TGE and PED divalence mouth Vaccine is taken, present invention uses one plant of probiotic excellent VREF obtained from the separation of chitling road colonic segment to resist as vaccine Original transmits live vector, is alternatively marked with substitute antibiotics resistance selective marker using enhanced green fluorescent protein (eGFP), Based on non-induced constitutive expression carrier pLXeno, constitutive expression TGEV S protein D antigen sites and PEDV S eggs are constructed White neutralizing epitope area ps420 restructuring VREF vaccine strain, realize that primary immune response prevents the purpose of two kinds of diseases, by the vaccine The oral vaccine that is prepared of strain is green, highly effective and safe, probiotic good.

On the basis of the studies above, the present invention proposes a kind of eGFP is marked, anti-swine infectious enterogastritis and pig stream The restructuring VREF vaccine strain of row diarrhoea, described vaccine strain are made with the VREF obtained from the separation of chitling road colonic segment Live vector is transmitted for vaccine antigen, wherein constitutive expression TGEV S protein D antigen sites and PEDV S containing eGFP marks The recombinant expression carrier of albumen neutralizing epitope area ps420 albumen.

Wherein, it is preferred that described VREF is named as VREF N-16 (Enterococcus Faecium N- 16), Classification And Nomenclature is VREF N-16 (Enterococcus Faecium N-16), is deposited in Chinese Typical Representative culture guarantor Tibetan center, in Chinese Wuhan Wuhan Universitys, its culture presevation numbering is CCTCC NO.M 2017516 for address, and the preservation time is On September 18th, 2017.

Wherein, it is preferred that containing successively with described in six of linker connections codings in described recombinant expression carrier The nucleotide sequence of TGEV S protein D antigens, the repetitive sequence are named as 6Ds, its nucleotide sequence such as SEQ ID NO.1 institutes Show, the nucleotide sequence of coding PEDV S protein neutralizing epitopes area ps420 albumen is as shown in SEQ ID NO.2.

Wherein, it is preferred that the constitutive expression TGEV S protein D antigen sites and PEDV S proteins of described eGFP marks The recombinant expression carrier of neutralizing epitope area ps420 albumen is transferred in the competent cell of VREF by electric method for transformation.

Wherein, it is preferred that the constitutive expression TGEV S protein D antigen sites and PEDV S proteins of described eGFP marks The recombinant expression carrier of neutralizing epitope area ps420 albumen is to be prepared by the following method to obtain：

(1) VREF constitutive expression carrier pLXeno structure

Transformed on the basis of plasmid pPG612, double digestion, enzyme are carried out to plasmid pPG612 using XbaI and XhoI Carrier segments after cutting are connected with eno-T7g10-PgsA anchor-MCS-rrnBT1T2 sequences, construct VREF composition Type expression plasmid, is named as pLXeno, and wherein eno is the constitutive promoter of lactic acid bacteria enolase, its nucleotide sequence such as SEQ Shown in ID NO.4；T7g10 is translational enhancer sequence, its nucleotide sequence as shown in SEQ ID NO.5, PgsAanchor's Nucleotide sequence is as shown in SEQ ID NO.6；MCS is polyclone enzyme enzyme site, restricted interior including at least Sac I and Apa I Enzyme cutting restriction enzyme site；RrnBT1T2 is Escherichia coli transcription terminator sequences, and its nucleotide sequence is as shown in SEQ ID NO.7；

(2) structure of 6Ds-ps420 fusion protein recombinant expression plasmids is expressed

Will expand obtain coding TGEV S protein D antigen sites, PEDV S protein neutralizing epitopes area's ps420 albumen with And eGFP nucleotide sequence is connected with plasmid pMD19-T simple, pMD18-T and pMD19-T simple respectively, is obtained Carrier is respectively designated as pMD19-Ts-6Ds, pMD18-T-ps420 and pMD19-Ts-eGFP；To recombinant plasmid pMD19- Ts-6Ds and pMD18-T-ps420 carries out double digestion, and the 6Ds genes of recovery purifying and pMD18-T-ps420 carrier segments are connected Connect, construction recombination plasmid pMD18-T-6Ds-ps420, using restriction endonuclease respectively to plasmid pMD18-T-6Ds- Ps420 and VREF constitutive expression carrier pLXeno carries out double digestion processing, through DNA glue reclaim kits purpose bases Because of fragment, the 6Ds-ps420 genetic fragments of purifying are connected with pLXeno fragments, structure recombinant expression plasmid pLXeno-6Ds- ps420；

(3) structure of eGFP marks expression 6Ds-ps420 fusion proteins restructuring VREF

Recombinant plasmid pMD19-Ts-eGFP and recombinant plasmid pLXeno-6Ds-ps420 is through double digestion, by recovery purifying EGFP genetic fragments be connected with the carrier segments of pLXeno-6Ds-ps420 mesh, construction recombination plasmid pLXeno-eGFP-6Ds- ps420；Double digestion processing is carried out to recombinant plasmid pLXeno-eGFP-6Ds-ps420 using restriction endonuclease, to cut Except chlorampenicol resistant encoding gene, through DNA glue reclaim kits target gene fragments, DNA Blunting Kit are utilized It is attached after the flat end of kit, builds the recombinant plasmid pLXeno-eGFP-6Ds-ps420 of non-resistance mark.

Further, the invention also provides a kind of method of the recombinant lactic acid bacteria vaccine strain described in structure, including it is following Step：

(1) VREF constitutive expression carrier pLXeno structure

Transformed on the basis of plasmid pPG612, double digestion, enzyme are carried out to plasmid pPG612 using XbaI and XhoI Carrier segments after cutting are connected with eno-T7g10-PgsA anchor-MCS-rrnBT1T2 sequences, construct VREF composition Type expression plasmid, is named as pLXeno, and wherein eno is the constitutive promoter of lactic acid bacteria enolase, its nucleotide sequence such as SEQ Shown in ID NO.4；T7g10 is translational enhancer sequence, its nucleotide sequence as shown in SEQ ID NO.5, PgsA anchor's Nucleotide sequence is as shown in SEQ ID NO.6；MCS is polyclone enzyme enzyme site, restricted interior including at least Sac I and Apa I Enzyme cutting restriction enzyme site；RrnBT1T2 is Escherichia coli transcription terminator sequences, and its nucleotide sequence is as shown in SEQ ID NO.7；

Recombinant plasmid pMD19-Ts-eGFP and recombinant plasmid pLXeno-6Ds-ps420 is through double digestion, by recovery purifying EGFP genetic fragments be connected with the carrier segments of pLXeno-6Ds-ps420 mesh, construction recombination plasmid pLXeno-eGFP- 6Ds-ps420, double digestion processing is carried out to recombinant plasmid pLXeno-eGFP-6Ds-ps420 using restriction endonuclease, To cut off chlorampenicol resistant encoding gene, through DNA glue reclaim kits target gene fragments, DNA Blunting are utilized It is attached after the flat end of Kit kits, builds the recombinant plasmid pLXeno-eGFP-6Ds-ps420 of non-resistance mark；

(4) electricity conversion

The recombinant plasmid pLXeno-eGFP-6Ds-ps420 electricity that step (3) structure obtains is transferred to VREF competence In cell, by recombinant bacterium of the flow cytometry screening with eGFP fluorescence labelings, passed so as to obtain pig that eGFP is marked, anti- The restructuring VREF of metachromia gastroenteritis and pig epidemic diarrhea.

In described method, it is preferred that described VREF is named as VREF N-16, is deposited in Chinese Typical Representative training Support thing collection, in Wuhan University, its culture presevation numbering is CCTCC NO.M 2017516 for address.

In described method, it is preferred that contain in the nucleotide sequence for the coding TGEV S protein D antigen sites for expanding to obtain Have successively with the nucleotide sequence of the described TGEV S protein D antigens of six codings of linker connections, repetitive sequence name For 6Ds, its nucleotide sequence is as shown in SEQ ID NO.1, the nucleotides of coding PEDV S protein neutralizing epitopes area ps420 albumen Sequence encodes eGFP nucleotide sequence as shown in SEQ ID NO.3 as shown in SEQ ID NO.2.

Further, the invention also provides described restructuring VREF vaccine strain is preparing prevention and treatment pig biography Purposes in metachromia gastroenteritis and pig epidemic diarrhea divalence oral vaccine.

A kind of transmissible gastroenteritis of swine and pig epidemic diarrhea divalence oral vaccine, it contains restructuring dung of the present invention Enterococcus vaccine strain.

Compared to prior art, the beneficial effects of the invention are as follows：

1st, compared with traditional inactivated vaccine and attenuated vaccine, the present invention is resisted by the use of probiotics VREF as vaccine is transmitted Former live vector develops anti-PEDV and TGEV divalence oral vaccine, and oral immunity can induce body to produce for mucosa infection the most Direct mucosal immune response, stimulate body to produce the sIgA antibody of sufficient amount, the protection to viral infection resisting is provided for body Power, immunization route are time saving and energy saving.Inactivated vaccine produced immunity time up to two weeks in pig body, and oral vaccine of the present invention is being exempted from It can detect within 3 days after epidemic disease specific sIgA in excrement, and VREF has the intestinal colonisation the characteristics of, can be in enteron aisle Continuous inducing producing specificity sIgA, the cause of disease of confrontation invasion in time, good immune effect, while can avoid using Attenuate vaccine Hidden danger.

2nd, VREF is safe and non-toxic as the main probiotics in probiotics, is widely used in food, feed adds Industry, there is the good colonization ability of inherent immunity adjuvant effect, mucoadhesive properties, animal intestinal tract and the enhancing non-spy of body The biological characteristicses such as specific immunological, as food grade expression vector can efficient expression alien gene, than attenuated pathogenic bacteria and disease Poison is safer as vaccine antigen transmission carrier.

3rd, inventor obtains an Enterococcus faecalis E.faecium N-16 strains from the separation of chitling road colonic segment, leads to Cross and 393 prebiotic performance comparisons of Lactobacillus casei L.casei, it was demonstrated that the VREF E.faecium N-16 strains are probiotic more It is good, the growth of delactational piglets can be remarkably promoted, improves the utilization rate of feed.The present invention uses VREF E.faecium N- 16 plants are used as vaccine antigen to transmit live vector, have developed anti-TGEV and PEDV infection restructuring VREF divalence oral vaccine, can Realize that primary immune response prevents the purpose of two kinds of diseases, there is the characteristics of green, highly effective and safe, probiotic good.

4th, the present invention instead of antibiotic resistance screening mark by the use of enhanced green fluorescence protein eGFP as selection markers Note, non-resistance mark restructuring VREF system is constructed, to prevent harm that the use of antibiotic-resistance marker is brought, makes it Product has more security, green.

5th, the present invention constructs expression PEDV S protein neutralizing epitopes area ps420 and TGEV using constitutive expression carrier The restructuring VREF oral formulations of S protein D antigen sites, vaccine antigen can constantly divide with the natural propagation of bacterial strain Secrete, and then effectively stimulate immune response, application prospect is considerable.

Brief description of the drawings

Fig. 1 is that delactational piglets feed the state observation after bacterium；

Fig. 2 is VREF constitutive expression carrier pLXeno Vector map；

Fig. 3 is recombinant plasmid pLXeno-6Ds-ps420 qualification results；

M：DNA marker；1：SacI digestion results；2：ApaI digestion results；3：SacI and ApaI double digestion results；4： Recombinant plasmid；

Fig. 4 is recombinant expression carrier pLXeno-eGFP-6Ds-ps420 qualification results；

M.DNA Marker 8000；2.pLXeno-eGFP-6Ds-ps420 is through SacI and ApaI double digestion results

Fig. 5 is colony PCR amplification result；

M.DNA Marker DL8000；1-21. target gene PCR amplifications .22：Blank control

Fig. 6 is the checking that restructuring VREF removes chloramphenicol resistance gene；

A：The flat boards of MRS containing chlorampenicol resistant；B：Without chlorampenicol resistant MRS flat boards；

1：Recombinate VREF pLXeno-6Ds-ps420/E.faecium N-16；2：Recombinate VREF pLXeno/E. faecium N-16；3：Recombinate VREF pLXeno-eGFP-6D-ps420/E.faecium N-16

Fig. 7 is the Western blot qualification results of purpose protein expression；

M：Pre-dyed albumen Marker；1：pLXeno-eGFP-6Ds-ps420/E.faecium N-16；2：pLXeno/E. faecium N-16

Fig. 8 is the fluorescence microscope testing result of eGFP expression；

A：pLXeno-eGFP-6Ds-ps420/E.faecium N-16；B：pLXeno/E.faecium N-16

Fig. 9 is immunized mice excrement moderate resistance TGEV and PEDV specificity sIgA antibody level testing results；

Figure 10 is immunized mice vaginal lotion moderate resistance TGEV and PEDV specificity sIgA antibody level testing results；

Figure 11 is that the anti-TGEV and PEDV IgG of specificity are horizontal in immune serum.

Culture presevation information：

Strain name：VREF N-16

Enterococcus Faecium N-16

Classification And Nomenclature：VREF N-16

Enterococcus Faecium N-16

Depositary institution：China typical culture collection center

Address：Chinese Wuhan Wuhan Universitys

Culture presevation is numbered： CCTCC NO.M 2017516

The preservation time：On September 18th, 2017

Embodiment

The present invention is further described with reference to specific embodiments and the drawings, advantages of the present invention and feature will be with Description and it is apparent.But embodiment is only exemplary, does not form any restrictions to the scope of the present invention.Art technology Personnel should be understood that without departing from the spirit and scope of the invention can be to the details and form of technical solution of the present invention Modify or replace, but these modifications and replacement are each fallen within protection scope of the present invention.

The VREF E.faecium N-16 strains of embodiment 1 and Lactobacillus casei L.casei 393 probiotic comparison

VREF E.faecium N-16 strains (CCTCC NO of the invention to being separated from chitling road:M 2017516) And structure genetic engineering lactic acid bacteria has often carried out comparative analysis with the probiotic of strain L. casei L.casei 393.Specifically Method is as follows：

Experiment pig is divided into 3 groups, and every group 10, the common of VREF E.faecium N-16 strains is added in experiment I group feedings Daily ration, experiment II group feeding addition Lactobacillus caseis L.casei 393 common daily ration, experiment III groups compare for common daily ration Group, after feeding 21 days, observe the growing state of piglet, it is seen that the delactational piglets of feeding VREF E.faecium N-16 groups More preferable compared with other two groups of entirety state of mind, hair is more smooth, bright, and skin is also more ruddy, and more love motion, alertness is high, Test result indicates that VREF E.faecium N-16 are added in daily ration can be obviously promoted the growth of piglet, its prebiotic effect Fruit is better than Lactobacillus casei L.casei 393, as a result sees Fig. 1.

Meanwhile by weighing the body weight of delactational piglets and the material weight of feeding, calculate the daily gain and material weight of every group of experiment pig Than as a result visible feeding VREF E.faecium N-16 groups are with feeding 393 groups of Lactobacillus casei L.casei and culture medium Control group compares the average daily gain for significantly improving delactational piglets, and significantly reduces feed-weight ratio (p≤0.05), as a result As shown in Tables 1 and 2.As can be seen here, feeding VREF E.faecium N-16 can significantly improve the growth of delactational piglets Performance, improve the utilization rate of feed.

The each group delactational piglets daily gain result of table 1

Note：Different capitalizations represent pole significant difference (p<0.01), different lowercase letter significant difference (p< 0.05)。

The each group delactational piglets feed-weight ratio result of table 2

The eGFP of embodiment 2 marks, amalgamation and expression TGEV 6Ds and PEDV ps420 restructuring VREFs structure

1st, the structure of pMD18-T-6Ds plasmids

The nucleotide sequence of TGEV TH98 strain D antigen sites (SCYTVSDSSFFSYGEIPFGVTDGPRYCY) will be encoded Repeat six times, linker sequences (GGCGGTGGCGGAGGCGGA) are inserted between every section, insert 5 linker, obtained weight altogether Complex sequences is named as 6Ds.6Ds gene orders are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, its nucleotides sequence Row are as shown in SEQ ID NO.1.Obtained gene order is connected with pMD18-T, converts TG1, recombinant bacterium extraction plasmid warp PCR, digestion and sequencing identification, recombinant plasmid are named as pMD18-T-6Ds.

2nd, the structure of pMD18-T-ps420 plasmids

PEDV strains are the PEDV HLJ-2012 strains that this laboratory is separately cultured.Utilize RNAFastl000 kits (Shanghai Fei Jie Reagent Companies) extracts viral RNA, reverse transcription cDNA, using cDNA as template amplification PEDV Neutralization and crystallizations area Gene ps420 (Accession No.：JX512907), the ps420 gene orders for expanding to obtain as shown in SEQ ID NO.2, It is connected with pMD18-T, converts TG1, recombinant bacterium extraction plasmid is named as pMD18- through PCR, digestion and sequencing identification, recombinant plasmid T-ps420。

3rd, the structure of pMD19-Ts-eGFP plasmids

Plasmid pEGFP-N1 (the Accession No. preserved with this laboratory：U55762.1) it is template amplification eGFP bases Cause, the eGFP gene orders for expanding to obtain are connected as shown in SEQ ID NO.3 with pMD18-T, convert TG1, recombinant bacterium extraction Plasmid is named as pMD18-T-eGFP through PCR, digestion and sequencing identification, recombinant plasmid.

4. encode the PCR amplifications of TGEV D locus genes, PEDV ps420 genes and eGFP genes

Using primers F s1 (restriction enzyme site containing SacI) and Rs1 (restriction enzyme site containing MluI), using pMD18-T-6Ds plasmids as Template, amplification 6Ds genetic fragments (594bp)；Using primers F s2 (restriction enzyme site containing MluI) and Rs2 (restriction enzyme site containing ApaI, And with flexible Linker coding gene sequences), using pMD18-T-ps420 plasmids as template, expand PEDV ps420 genes (420bp)；Utilize primers F e (restriction enzyme site containing SacI) and Re (restriction enzyme site containing KpnI, and base is encoded with flexible Linker Because of sequence), using pMD18-T-eGFP plasmids as template, expand enhanced green fluorescence protein eGFP genes (717bp).Primer sequence Row are shown in Table 3.

The primer sequence of table 3

5. express the structure of 6Ds-ps420 and eGFP-6Ds-ps420 fusion proteins restructuring VREF

5.1 VREF constitutive expression carrier pLXeno structure

Transformed on the basis of plasmid pPG612, double digestion, enzyme are carried out to plasmid pPG612 using XbaI and XhoI Carrier segments after cutting are connected with eno-T7g10-PgsA anchor-MCS-rrnBT1T2 sequences, construct VREF composition Type expression plasmid, is named as pLXeno, and wherein eno is the constitutive promoter of lactic acid bacteria enolase, its nucleotide sequence such as SEQ Shown in ID NO.4；T7g10 is translational enhancer sequence, its nucleotide sequence as shown in SEQ ID NO.5, PgsA anchor's Nucleotide sequence is as shown in SEQ ID NO.6；MCS is polyclone enzyme enzyme site；RrnBT1T2 is Escherichia coli transcription terminator Sequence, its nucleotide sequence is as shown in SEQ ID NO.7；Vector map is as shown in Figure 2.

The structure of 5.2 expression 6Ds-ps420 fusion protein restructuring VREFs

Gene 6Ds, ps420 and eGFP that above-mentioned amplification is obtained respectively with plasmid pMD19-T simple (referred to as PMD19-Ts), pMD18-T with pMD19-T simple are connected, and MluI and ApaI is carried out to recombinant plasmid pMD18-T-ps420 Double digestion, MluI and SacI double digestions, ApaI and SacI double digestions, PCR and sequencing identification.Utilize SacI and ApaI double digestion weights Group plasmid pMD19-Ts-6Ds and pMD18-T-ps420, through DNA glue reclaim kits target gene fragments, will be reclaimed pure The 6Ds genes of change are connected with pMD18-T-ps420 carrier segments, construction recombination plasmid pMD18-T-6Ds-ps420.Utilize limitation Property endonuclease SacI and ApaI are respectively to plasmid pMD18-T-6Ds-ps420 and VREF constitutive expression carrier PLXeno carries out double digestion processing, through DNA glue reclaim kits target gene fragments, by the 6Ds-ps420 genes of purifying Fragment is connected with pLXeno fragments, builds recombinant expression plasmid pLXeno-6Ds-ps420, the digestion qualification result of recombinant plasmid See Fig. 3.Prepare VREF E.faecium N-16 (CCTCC NO:M 2017516) competent cell, will restructuring through electricity conversion Plasmid pLXeno-6Ds-ps420 is transferred to E.faecium N-16, is screened in the Selective agar medium containing chlorampenicol resistant positive Clone, is named as pLXeno-6Ds-ps420/E.faecium N-16.

The structure of 5.3eGFP mark expression 6Ds-ps420 fusion protein restructuring VREFs

For recombinant plasmid pMD19-Ts-eGFP through SacI and KpnI double digestions, recovery size is about 750bp target gene eGFP；For recombinant plasmid pLXeno-6Ds-ps420 through SacI and KpnI double digestions, recovery size is about the carrier-pellet of 6000bp mesh Section.The eGFP genetic fragments of recovery purifying are connected with the carrier segments of pLXeno-6Ds-ps420 mesh, construction recombination plasmid PLXeno-eGFP-6Ds-ps420, digestion qualification result are shown in Fig. 4.Using restriction endonuclease StuI and NcoI to restructuring Plasmid pLXeno-eGFP-6Ds-ps420 carries out double digestion processing, to cut off chlorampenicol resistant encoding gene, through DNA glue reclaims Kits target gene fragment, using being attached after the flat end of DNA Blunting Kit kits, structure is non-anti- Property mark recombinant plasmid pLXeno-eGFP-6Ds-ps420.

Prepare VREF E.faecium N-16 (CCTCC NO:M 2017516) competent cell, through electric transformation technology Recombinant plasmid pLXeno-eGFP-6Ds-ps420 is transferred in E.faecium N-16, through FACS Calibur flow cytometers In recombinant bacterium of the wavelength 488nm screenings with green florescent signal, and prepare 24 orifice plates, flow cytometry has been filtered out into fluorescence The bacterium of signal is transferred in 24 orifice plates, per 20, hole bacterium, is then coated on the MRS agar plates of non-resistant, 37 DEG C of cultures 36h.Picking single bacterium colony enters performing PCR identification, using Fs1 and Re as both sides primer, passes through colony PCR amplification target gene eGFP, bacterium Fall PCR qualification results and see Fig. 5, the restructuring VREF for identifying positive is named as pLXeno-eGFP-6Ds-ps420/ E.faecium N-16。

Recombinate VREF and remove chlorampenicol resistant checking：By the weight of the non-resistance mark being incubated overnight (eGFP marks) Group VREF pLXeno-eGFP-6Ds-ps420/E.faecium N-16, restructuring VREF pLXeno-6Ds-ps420/ E.faecium N-16 and restructuring VREF pLXeno/E.faecium N-16 are respectively in the MRS agar containing chlorampenicol resistant Rule on flat board and MRS agar plates without chlorampenicol resistant culture, 37 DEG C of quiescent culture 36h.As a result as shown in Figure 6, eGFP (non-resistance mark) restructuring VREF pLXeno-eGFP-6Ds-ps420/E.faecium N-16 are marked to be trained in nonreactive MRS Normal growth on base is supported, is not grown on the MRS agar mediums containing chlorampenicol resistant, and compares restructuring VREF PLXeno-6Ds-ps420/E. faecium N-16 and pLXeno/E.faecium N-16 train in nonreactive and MRS containing chloramphenicol Support equal normal growth in base.

6. the expression and identification of destination protein

6.1Western blot are identified

37 DEG C of quiescent cultures recombinate VREF pLXeno-eGFP-6Ds-ps420/E.faecium N-16 16h, collect Thalline simultaneously cracks, and mycoprotein is transferred to pvdf membrane after SDS-PAGE electrophoresis, using rabbit-anti ps420 albumen hyper-immune serum as primary antibody Western blot detections are carried out, as a result as shown in Figure 7, destination protein is expressed, and albumen size is consistent with expection.

6.2 fluorescence microscope

Picking pLXeno-eGFP-6Ds-ps420/E.faecium N-16 single bacterium colonies are inoculated in 5mL MRS fluid nutrient mediums In, 37 DEG C of culture 16h；Take 1mL bacterium solutions 5000rpm to centrifuge 5min, abandon supernatant, wash thalline 3 times with sterile PBS buffer, thalline Precipitation PBS hangs, and takes 10 μ L smears, the expression through fluorescence microscope eGFP.As a result as shown in Figure 8, The visible obvious green fluorescence (A) of VREF pLXeno-eGFP-6Ds-ps420/E. faecium N-16 thalline is recombinated, and Control group VREF pLXeno/E.faecium N-16 thalline has no green fluorescence.

Embodiment 3 recombinates VREF pLXeno-eGFP-6Ds-ps420/E.faecium N-16 Evaluation of Immunogenicity

The present invention have rated the restructuring VREF pLXeno-eGFP- by animal model system of BALB/c mouse 6Ds-ps420/E.faecium N-16 immunogenicity.8 week old BALB/c mouses are divided into 4 groups, and every group 10, difference is by oral administration Approach inoculation restructuring VREF pLXeno-eGFP-6Ds-ps420/E.faecium N-16, pLXeno-6Ds-ps420/ E.faecium N-16 and pLXeno/E.faecium N-16, oral dose are 200 μ L 2 × 10⁹CFU/mL recombinates dung intestines ball Bacterium, oral 200 μ L PBS are control.Immune programme for children is every 2 weeks immune 1 time, is immunized 3 times altogether.Detected using ELISA method IgG antibody is horizontal in sIgA antibody levels and serum in BALB/c mouse excrement, vaginal mucus, the detection of antibody neutralization titer And lymphocyte proliferation assay.Concrete outcome is as follows.

The measure of 1 specific sIgA antibody

Specific sIgA antibody test results in 1.1 immunized mice excrement

Respectively at immune preceding and each immune rear 1d, 2d, 7d collection stool in mice sample, detected using ELISA method Anti- TGEV and PEDV specificity sIgA antibody levels.As shown in Figure 9, oral immunity recombinates VREF pLXeno- to testing result Inducing mouse body after eGFP-6Ds-ps420/E.faecium N-16 and pLXeno-6Ds-ps420/E. faecium N-16 Generate significantly in anti-TGEV and the PEDV specificity of control group (PBS groups and pLXeno/E.faecium N-16 empty carriers group) SIgA antibody levels, it is not notable that group difference is immunized in two restructuring VREFs.

Specific sIgA TPPAs in 1.2 immunized mice vaginal lotions

Before immune and rear 7d is immunized every time and collects mouse vagina washing lotion sample, anti-TGEV is detected using ELISA method It is horizontal with PEDV specialized mucosal antibody sIgA.As shown in Figure 10, oral immunity recombinates VREF pLXeno- to testing result Inducing mouse body after eGFP-6Ds-ps420/E.faecium N-16 and pLXeno-6Ds-ps420/E.faecium N-16 Generate significantly in anti-TGEV and the PEDV specificity of control group (PBS groups and pLXeno/E.faecium N-16 empty carriers group) SIgA antibody levels, it is not notable that group difference is immunized in two restructuring VREFs.

Specific antibody IgG is determined in 2 immune serums

Respectively using TGEV and PEDV totivirus as the hole ELISA reaction plates of antigen coat 96, with the immune restructuring dung of collection Enterococcus mice serum is primary antibody, and HRP mark sheep anti-mouse iggs are secondary antibody, and detection immune serum moderate resistance TGEV and PEDV are special Heterogenetic antibody IgG is horizontal.As shown in Figure 11, restructuring VREF pLXeno-eGFP-6Ds-ps420/ is immunized in testing result Inducing mouse body is generated significantly in right after E.faecium N-16 and pLXeno-6Ds-ps420/E.faecium N-16 According to the anti-TGEV and PEDV specific serums IgG antibody water of group (PBS groups and pLXeno/E.faecium N-16 empty carriers group) Flat, it is not notable that group difference is immunized in two restructuring VREFs.

3 antibody neutralization titers detect

TGEV and PEDV are made to 10 times of progressive dilutions i.e. 10 on 96 well culture plates respectively^-1, 10^-2, 10^-3..., the disease per hole Malicious suspension amount is 100 μ L, and each dilution factor makees 8 holes, adds 100 μ L ST or Vero cell suspensions per hole, culture plate last Row is set to cell controls (concentration of cell suspension covers with individual layer as degree using cell in 24h), is placed in the 37 DEG C of trainings of 5%CO2 incubators Support, observe cytopathy day by day.ST cells start obvious lesion occur for second day after meeting TGEV, and Vero cells are connect after PEDV Start within three days obvious lesion occur, TGEV TCID are calculated by Reed and Muench Liang Shi methods₅₀About 10^-4.83, PEDV TCID₅₀For 10^-3.83。

The serum antibody obtained using the method for fixed virus dilution antibody to the oral immune mouse of restructuring VREF Carry out the measure of viral neutralization titer.It is 100TCID per hole virus concentration₅₀, serum antibody is with 2 times of progressive dilutions, i.e., and 1：10,1： 20,1：40,1：80,1：160 ..., after virus mixes with the antibody after dilution, 37 DEG C of incubation 1h, then access ST or Vero Cell, culture plate is put into 5%CO₂, 37 DEG C of constant incubator cultures, observe cytopathy day by day and record result, counted Analysis.Testing result shows that in immune mouse serum antibody and TGEV potency is 1：100, it is 1 to neutralize PEDV potency：112.2.

4 spleen lymphocyte proliferations are tested

4.1TGEV 6Ds proteantigens stimulate spleen lymphocyte proliferation

It is sterile to take immune mouse spleen cell in vitro culture, respectively with final concentration of 1 μ g/mL, 5 μ g/mL and 25 μ g/mL TGEV 6Ds antigenic stimulus, as a result show (being shown in Table 4)：5 μ g groups are to the proliferation function highest of splenocyte, and 25 μ g groups are to splenocyte Proliferation function be higher than 1 μ g groups (p<0.05), but less than 5 μ g groups to the proliferation function of splenocyte.

The various concentrations 6Ds of table 4 stimulates the proliferation function of splenocyte

4.2PEDV ps420 proteantigens stimulate spleen lymphocyte proliferation

It is sterile to take immune mouse spleen cell in vitro culture, respectively with final concentration of 1 μ g/mL, 5 μ g/mL and 25 μ g/mL PEDV ps420 antigenic stimulus, as a result show (being shown in Table 5)：1 μ g groups and 5 μ g groups are higher than 25 μ g groups (p to the proliferation function of splenocyte <0.05)

The various concentrations ps420 of table 5 stimulates the proliferation function of splenocyte

In summary, the present invention uses the VREF E.faecium N-16 strains separated from chitling road to resist as vaccine Anti- TGEV and PEDV restructuring VREF the divalence oral vaccine that original transmits carrier development effectively induced animal body can produce Significantly mucosa-immune reaction and systemic immune reaction, it is shown that good immunogenicity, and with well probiotic.

Sequence table

<110>Northeast Agricultural University

<120>A kind of eGFP mark, anti-swine infectious enterogastritis and pig epidemic diarrhea restructuring VREF vaccine strain and It is applied

<130> klpi170707

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 594

<212> DNA

<213> 6Ds

<400> 1

tcttgttata ctgtttcaga ctcatcattt ttctcatatg gtgaaattcc atttggcgtt 60

actgatggtc cacggtattg ttatggcggt ggcggaggcg gatcttgtta tactgtttca 120

gactcatcat ttttctcata tggtgaaatt ccatttggcg ttactgatgg tccacggtat 180

tgttatggcg gtggcggagg cggatcttgt tatactgttt cagactcatc atttttctca 240

tatggtgaaa ttccatttgg cgttactgat ggtccacggt attgttatgg cggtggcgga 300

ggcggatctt gttatactgt ttcagactca tcatttttct catatggtga aattccattt 360

ggcgttactg atggtccacg gtattgttat ggcggtggcg gaggcggatc ttgttatact 420

gtttcagact catcattttt ctcatatggt gaaattccat ttggcgttac tgatggtcca 480

cggtattgtt atggcggtgg cggaggcgga tcttgttata ctgtttcaga ctcatcattt 540

ttctcatatg gtgaaattcc atttggcgtt actgatggtc cacggtattg ttat 594

<210> 2

<211> 420

<212> DNA

<213> ps420

<400> 2

gttactttgc catcatttaa tgatcattct tttgttaaca ttactgtctc tgcgtccttt 60

ggtggtcata gtggtgccaa ccttattgca tctgacacta ctatcaatgg gtttagttct 120

ttctgtgttg acactagaca atttaccatt tcactgtttt ataacgttac aaacagttat 180

ggttatgtgt ctaaatcaca ggacagtaat tgccctttca ccttgcaatc tgttaatgat 240

tacctgtctt ttagtaaatt ttgtgtttcc accagccttt tggctagtgc ctgtaccata 300

gatctttttg gttaccctga gtttggtagt ggtgttaagt ttacgtccct ttactttcaa 360

ttcacaaagg gtgagttgat tactggcacg cctaaaccac ttgaaggtgt cacggacgtt 420

<210> 3

<211> 717

<212> DNA

<213> eGFP

<400> 3

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717

<210> 4

<211> 776

<212> DNA

<213> eno

<400> 4

gcatattaca aaaaagtcct ctgctcgcta acgcgggtgg agggcttatt tttttgaaac 60

taagaattca cttttcgtta ctgtgcaatt tgtgtgcaat ttggtccttt aataaccgct 120

acataagtgt ttcgtactcc cgggctgcgc agaaacccgg ttaaatggga aaaggtagga 180

cagcataacc ccgagtaatc ggggtttttg ttttgtttaa aattaaaaac attcaaaaca 240

ggatagaggc gtgacatgca tcatgctaaa acgccgataa gtgatagcct gagatccgaa 300

ttgcagcgac gatcttcacc gttcattgct cgggtgcttg atgagagatt cggcggctat 360

ttttgcaaac gctaacatta tgacttggaa actgctcatt atataacgaa cgggaataaa 420

accaaccctt gtcactactg aactaagaca caaactgaat cacttttccc agcatgaaaa 480

ttccattaga aacaacccga aacccgcttt caaaaaaagc cgccagacgg gatgatgttt 540

tagtaaaatc cgaataatct tcacaccaaa aaaatagatg ttctaagttt accgaatatg 600

tgattgtttt tggacgatta aggccgcaga cttgcgataa agcggaaccc ctgctacaat 660

tggtcttgtt ggtaaaggaa aacagcattt gttgggttcc taattttttt cagcttgctg 720

ggtcggcgaa cgactcggag ggacgtgaaa tgacccatta aagaaggacg tgcctt 776

<210> 5

<211> 22

<212> DNA

<213> T7g10

<400> 5

aataattttg tttaacttta ag 22

<210> 6

<211> 1143

<212> DNA

<213> PgsA anchor

<400> 6

atgaaaaaag aactgagctt tcatgaaaag ctgctaaagc tgacaaaaca gcaaaaaaag 60

aaaaccaata agcacgtatt tattgccatt ccgatcgttt ttgtccttat gttcgctttc 120

atgtgggcgg gaaaagcgga aacgccgaag gtcaaaacgt actctgacga cgtactctca 180

gcctcatttg taggcgatat tatgatggga cgctatgttg aaaaagtaac ggagcaaaaa 240

ggggcagaca gtatttttca atatgttgaa ccgatcttta gagcctcgga ttatgtagca 300

ggaaactttg aaaacccggt aacctatcaa aagaattata aacaagcaga taaagagatt 360

catctgcaga cgaataagga atcagtgaaa gtcttgaagg atatgaatct cacggttctc 420

aacagcgcaa acaaccacgc aatggattac ggcgttcagg gcatgaaaga tacgcttgga 480

gaatttgcga agcaaaacct tgatatcgtt ggagcgggat acagcttaag tgatgcgaaa 540

aagaaaattt tgtaccagaa agtcaacggg gtaacgattg caacgcttgg ctttaccgat 600

gtgtccggga aaggtttcgc ggctaaaaag aatacgccgg gcgtgctgcc cgcagatcct 660

gaaatcttca tccctatgat ttcagaagcg aaaaaacatg ctgacattgt tgttgtgcag 720

tcacactggg gacaagagta tgacaatgat ccaaatgacc gccagcgcca gcttgcaaga 780

gccatgtctg atgcgggagc tgacatcatc gtcggccatc acccgcacat cttagaaccg 840

attgaagtat ataacggaac cgtcattttc tacagcctcg gcaactttgt ctttgatcaa 900

ggctggacga gaacaagaga cagtgcactg gttcagtatc acctgaagaa aaatggaaca 960

ggccgctttg aagtgacacc gatcgatatc catgaagcga cacctgcacc tgtgaaaaaa 1020

gacagcctta aacagaaaac cattattcgc gaactgacga aagactctaa tttcgcttgg 1080

aaagtagaag acggaaaact gacgtttgat attgatcata gtgacaaact aaaatctaaa 1140

taa 1143

<210> 7

<211> 404

<212> DNA

<213> rrnBT1T2

<400> 7

ttggcttatg agagaagatt ttcagcctga tacagattaa atcagaacgc agaagcggtc 60

tgataaaaca gaatttgcct cccggcagta gcgcggtggt cccacctgac cccatgccga 120

actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat gcgagagtag 180

ccaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt 240

atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg 300

aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg 360

catcaaagga atcagaaggc catcctgacg gatggccttt ttgc 404

Claims

1. the restructuring VREF vaccine strain of a kind of eGFP is marked, anti-swine infectious enterogastritis and pig epidemic diarrhea, it is special Sign is that described vaccine strain transmits live vector using the VREF obtained from the separation of chitling road colonic segment as vaccine antigen, Constitutive expression TGEV S protein D antigen sites and PEDV S protein neutralizing epitopes area ps420 eggs wherein containing eGFP marks White recombinant expression carrier.

2. restructuring VREF vaccine strain as claimed in claim 1, it is characterised in that described VREF is named as dung intestines Coccus N-16, is deposited in China typical culture collection center, and in Wuhan University, its culture presevation numbering is CCTCC for address NO.M 2017516。

3. restructuring VREF vaccine strain as claimed in claim 1, it is characterised in that contain in described recombinant expression carrier It is named as successively with the nucleotide sequence of the described TGEV S protein D antigens of six codings of linker connections, the repetitive sequence 6Ds, its nucleotide sequence is as shown in SEQ ID NO.1, the nucleotides sequence of coding PEDV S protein neutralizing epitopes area ps420 albumen Row are as shown in SEQ ID NO.2.

4. restructuring VREF vaccine strain as claimed in claim 1, it is characterised in that the composing type table of described eGFP marks Converted up to the recombinant expression carrier of TGEV S protein D antigen sites and PEDV S protein neutralizing epitopes area ps420 albumen by electricity Method is transferred in the competent cell of VREF.

5. restructuring VREF vaccine strain as claimed in claim 1, it is characterised in that the composing type table of described eGFP marks Recombinant expression carrier up to TGEV S protein D antigen sites and PEDV S protein neutralizing epitopes area ps420 albumen is by following Method is prepared：

(1) VREF constitutive expression carrier pLXeno structure

Transformed on the basis of plasmid pPG612, double digestion is carried out to plasmid pPG612 using XbaI and XhoI, after digestion Carrier segments be connected with eno-T7g10-PgsAanchor-MCS-rrnBT1T2 sequences, construct VREF composing type table Up to plasmid, pLXeno is named as, wherein eno is the constitutive promoter of lactic acid bacteria enolase, its nucleotide sequence such as SEQ ID Shown in NO.4；T7g10 is translational enhancer sequence, and its nucleotide sequence is as shown in SEQ ID NO.5, PgsA anchor core Nucleotide sequence is as shown in SEQ ID NO.6；MCS is polyclone enzyme enzyme site, including at least Sac I and Apa I restriction enzymes Enzyme restriction enzyme site；RrnBT1T2 is Escherichia coli transcription terminator sequences, and its nucleotide sequence is as shown in SEQ ID NO.7；

Obtained coding TGEV S protein D antigen sites, PEDV S protein neutralizing epitopes area's ps420 albumen and eGFP will be expanded Nucleotide sequence be connected respectively with plasmid pMD19-T simple, pMD18-T and pMD19-T simple, obtained carrier point PMD19-Ts-6Ds, pMD18-T-ps420 and pMD19-Ts-eGFP are not named as it；To recombinant plasmid pMD19-Ts-6Ds and PMD18-T-ps420 carries out double digestion, and the 6Ds genes of recovery purifying are connected with pMD18-T-ps420 carrier segments, structure weight Group plasmid pMD18-T-6Ds-ps420, using restriction endonuclease respectively to plasmid pMD18-T-6Ds-ps420 and dung intestines Coccus constitutive expression carrier pLXeno carries out double digestion processing, will be pure through DNA glue reclaim kits target gene fragments The 6Ds-ps420 genetic fragments of change are connected with pLXeno fragments, structure recombinant expression plasmid pLXeno-6Ds-ps420；

Recombinant plasmid pMD19-Ts-eGFP and recombinant plasmid pLXeno-6Ds-ps420 is through double digestion, by recovery purifying EGFP genetic fragments are connected with the carrier segments of pLXeno-6Ds-ps420 mesh, construction recombination plasmid pLXeno-eGFP-6Ds- Ps420, double digestion processing is carried out to recombinant plasmid pLXeno-eGFP-6Ds-ps420 using restriction endonuclease, to cut Except chlorampenicol resistant encoding gene, through DNA glue reclaim kits target gene fragments, tried using DNA Blunting Kit It is attached after the flat end of agent box, builds the recombinant plasmid pLXeno-eGFP-6Ds-ps420 of non-resistance mark.

6. it is a kind of build claim any one of 1-5 described in recombinant lactic acid bacteria vaccine strain method, it is characterised in that including with Lower step：

(1) VREF constitutive expression carrier pLXeno structure

Transformed on the basis of plasmid pPG612, double digestion is carried out to plasmid pPG612 using XbaI and XhoI, after digestion Carrier segments be connected with eno-T7g10-PgsA anchor-MCS-rrnBT1T2 sequences, construct VREF composing type table Up to plasmid, pLXeno is named as, wherein eno is the constitutive promoter of lactic acid bacteria enolase, its nucleotide sequence such as SEQ ID Shown in NO.4；T7g10 is translational enhancer sequence, and its nucleotide sequence is as shown in SEQ ID NO.5, PgsA anchor core Nucleotide sequence is as shown in SEQ ID NO.6；MCS is polyclone enzyme enzyme site, including at least Sac I and Apa I restriction enzymes Enzyme restriction enzyme site；RrnBT1T2 is Escherichia coli transcription terminator sequences, and its nucleotide sequence is as shown in SEQ ID NO.7；

Recombinant plasmid pMD19-Ts-eGFP and recombinant plasmid pLXeno-6Ds-ps420 is through double digestion, by recovery purifying EGFP genetic fragments are connected with the carrier segments of pLXeno-6Ds-ps420 mesh, construction recombination plasmid pLXeno-eGFP-6Ds- Ps420, double digestion processing is carried out to recombinant plasmid pLXeno-eGFP-6Ds-ps420 using restriction endonuclease, to cut Except chlorampenicol resistant encoding gene, through DNA glue reclaim kits target gene fragments, tried using DNA Blunting Kit It is attached after the flat end of agent box, builds the recombinant plasmid pLXeno-eGFP-6Ds-ps420 of non-resistance mark；

(4) electricity conversion

The recombinant plasmid pLXeno-eGFP-6Ds-ps420 electricity that step (3) structure obtains is transferred to VREF competent cell In, by recombinant bacterium of the flow cytometry screening with eGFP fluorescence labelings, so as to obtain stomach that eGFP is marked, anti-swine infectious The restructuring VREF of enteritis and pig epidemic diarrhea.

7. method as claimed in claim 6, it is characterised in that described VREF is named as VREF N-16, preservation In China typical culture collection center, in Wuhan University, its culture presevation numbering is CCTCC NO.M 2017516 for address.

8. method as claimed in claim 6, it is characterised in that expand the obtained core of coding TGEV S protein D antigen sites Contain in nucleotide sequence successively with the nucleotide sequences of the described TGEV S protein D antigens of six codings of linker connections, should Repetitive sequence is named as 6Ds, and its nucleotide sequence encodes PEDV S protein neutralizing epitopes area ps420 as shown in SEQ ID NO.1 The nucleotide sequence of albumen encodes eGFP nucleotide sequence as shown in SEQ ID NO.3 as shown in SEQ ID NO.2.

9. the restructuring VREF vaccine strain described in claim any one of 1-5 is preparing prevention and treatment transmissible gastroenteritis of swine With the purposes in pig epidemic diarrhea divalence oral vaccine.

10. a kind of transmissible gastroenteritis of swine and pig epidemic diarrhea divalence oral vaccine, it is characterised in that contain claim 1- Restructuring VREF vaccine strain described in 5 any one.